Spatiotemporal transcriptomic maps of whole mouse embryos at the onset of organogenesis

Spatiotemporal orchestration of gene expression is required for proper embryonic development. The use of single-cell technologies has begun to provide improved resolution of early regulatory dynamics, including detailed molecular definitions of most cell states during mouse embryogenesis. Here we used Slide-seq to build spatial transcriptomic maps of complete embryonic day (E) 8.5 and E9.0, and partial E9.5 embryos. To support their utility, we developed sc3D, a tool for reconstructing and exploring three-dimensional ‘virtual embryos’, which enables the quantitative investigation of regionalized gene expression patterns. Our measurements along the main embryonic axes of the developing neural tube revealed several previously unannotated genes with distinct spatial patterns. We also characterized the conflicting transcriptional identity of ‘ectopic’ neural tubes that emerge in Tbx6 mutant embryos. Taken together, we present an experimental and computational framework for the spatiotemporal investigation of whole embryonic structures and mutant phenotypes

Identifizierung von Protogenin als neuartiger Oberflächenmarker für frühe kortikale neurale Stammzellen

During mammalian corticogenesis, a wide diversity of neural stem cells (NSCs) orchestrate the development and organization of the cortex. The pool of NSCs initially expands through proliferative symmetric divisions, and sequentially starts dividing asymmetrically to give rise to the diverse cell types residing within the cortical layers. Throughout this process, cortical NSCs undergo extensive modifications in their transcriptomic profile and chromatin landscape contributing to the formation of heterogeneous progenitor populations. Although much progress has been made towards understanding cell-fate specification during human corticogenesis the mechanisms responsible for the temporal lineage specification of NSCs remain largely unknown. Understanding the variability of these distinct NSC populations is key for developing an in vitro system that allows for the homogeneous and unlimited culture of the desired NSC type which is crucial for cell replacement-based therapies. Hence, one of the main aims in our lab is to identify and discern these distinct NSC types which sequentially appear during cortical development with the objective to better understand these cell stages and, eventually, being able to manipulate them in vitro. In order to address this question, my project is focused on developing a strategy to isolate the early cortical NSC population for its characterization and potential manipulation. The main approach is to identify a cell surface marker to enable the isolation of these cells from our in vitro culture by fluorescence-activated cell sorting. By profiling our hiPSC-derived cortical progenitors at different stages by means of single-cell RNA sequencing, we selected potential candidate markers that were validated using immunofluorescence and sequencing methods. In this study, we identify Protogenin (PRTG) as a novel surface marker for early human cortical NSCs that can be used to isolate this population in vitro. We provide evidence that early expression of the novel marker correlates with cortical lineage specification. Furthermore, by sorting for such marker at early stages of neural induction we can prospectively isolate three distinct cortical subpopulations, resulting in highly pure subtype-specific NSC cultures. These findings illustrate the utility of PRTG cell-surface sorting for enriching early cortical NSCs in culture and, thus, aiding to develop a more robust and homogenous differentiation protocol. Ultimately, such knowledge should facilitate the generation of highly pure stage- and region-specific NSC populations from patient-derived samples which would provide a reliable source for cell replacement and regenerative therapies

Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling